Summary

In vitro metabolism of perospirone was examined with rat, monkey and human liver S9, human liver microsomes and yeast microsomes expressing human P450, using14C labeled perospirone. With rat liver S9, the major metabolites were MX9 and ID-11614, produced by cleavage at the butylene chain. However, some butylene non-cleavage and hydration of the cyclohexane ring were found, although limited in extent. Unknown metabolites accounted for about 10% of the total. After incubation for 10 minutes with monkey liver S9, the major metabolites were ID-15036 and MX11, hydrated in the cyclohexane ring. After incubation for 60 minutes, ID-15001, i.e. the butylene chain cleavage type increased. Unknown metabolites accounted for about 20%. After incubation for 10 minutes with human liver S9, the major metabolite was ID-15036, hydrated in the cyclohexane ring. In addition, MX11 and many unknown metabolites were evident. After incubation for 60 minutes, the butylene chain cleavage type and unknown metabolites increased. Individual differences were found in the metabolic reaction rate. With human liver microsomes, MX11, ID-15001 and unknown metabolites were again the major metabolites. With yeast microsomes expressing human P450 subtypes, CYP1A1, 2C8, 2D6, 3A4 were responsible for the metabolism in particular, and CYP3A4 contributes greatly. Therefore it is unlikely that genetic polymorphism will arise a present a problem with regard to the clinical drug.

The results demonstrated that the main metabolic pathway in human liver S9 and liver microsomes involve oxidation at cyclohexane, oxidative cleavage of the butylene side chain and S-oxidation. The same was the case in rat and monkey S9, but species differences were found in the proportions of the metabolites produced.

Sirtuins are recently discovered NAD+-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins, thereby regulating the biological function of their targets. Sirtuins have been shown to increase organism and tissue survival in diverse organisms, ranging from yeast to mammals. Evidence indicates that NAD+ metabolism and sirtuins contribute to mechanisms that influence cell survival under conditions of stress and toxicity. For example, recent work has shown that sirtuins and increased NAD+ biosynthesis provide protection against neuron axonal degeneration initiated by genotoxicity or trauma. In light of their protective effects, sirtuins and NAD+ metabolism could represent therapeutic targets for treatment of acute and chronic neurodegenerative conditions. Our work has focused on elucidating the enzymatic functions of sirtuins and quantifying perturbations of cellular NAD+ metabolism. We have developed mass spectrometry methods to quantitate cellular NAD+ and nicotinamide. These methods allow the quantitation of changes in the amounts of these metabolites in cells caused by chemical and genetic interventions. Characterization of the biochemical properties of sirtuins and investigations of NAD+ metabolism are likely to provide new insights into mechanisms by which NAD+ metabolism regulates sirtuin activities in cells. To develop new strategies to improve cell stress resistance, we have initiated proof of concept studies on pharmacological approaches that target sirtuins and NAD+ metabolism, with the goal of enhancing cell protection against genotoxicity.

The toxicokinetics of pentachlorophenol (PCP) were studied in B6C3F1 mice, a strain in which PCP was previously found to be carcinogenic. In a crossover design, doses of 15 mg/kg were given intravenously (bolus) and orally (gastric intubation) to six animals. Concentrations of PCP in blood, urine, and feces were measured by capillary gas chromatography with electron-capture detection. After intravenous administration, the values of clearance and volume of distribution were 0.057 ± 0.007 L/hr/kg and 0.43 ± 0.06 L/kg, respectively. These two parameters exhibited low intermouse variability (coefficients of variation <14%). The elimination half-life was 5.2 ± 0.6 hr. After oral administration, the PCP peak plasma concentration (28 ± 7 µg/ml) occurred at 1.5 ± 0.5 hr and absorption was complete (bioavailability = 1.06 ± 0.09). The elimination half-life was 5.8 ± 0.6 hr. Only 8% of the PCP dose was excreted unchanged by the kidney. PCP was primarily recovered in urine as conjugates. A portion of the dose was recovered in urine as the mutagen, tetrachlorohydroquinone (5%) (TCHQ), and its conjugates (15%). For both PCP and TCHQ, sulfates accounted for 90% or more of the total conjugates (glucuronides and sulfates).

Summary

11 patients (9m, 2f, median age 59 years) with ventricular ectopic activity of at least Lown grade III received 20 mg N-Propylajmaline-bitartrate (N-PAB)* p.o. Plasma concentrations of N-PAB were determined with HPLC from blood samples within 26 hours after administration. An open two-compartment model was used. In 8 patients with normal function of the liver and the kidneys, the median clearance of N-PAB was 6.86 ml/min/kg and the median volume of distribution was 1.56 1/kg. Two patients had a clearly diminished clearance of 1.58 ml/min/kg without obvious impairment of liver or renal function. One patient with chronic glomerulonephritis (plasma creatinine 3.4 mg/dl) had a N-PAB clearance of 2.79 ml/min/kg. None of the Spearman rank correlation coefficients between the pharmacokinetic parameters of N-PAB with age, plasma albumin, albumin/globulin-quotient, plasma creatinine and cholin-esterase were significant. All calculated parameters were in the range determined in young subjects. It is concluded that physiological changes with age do not lead to significant changes of the pharmacokinetics of N-PAB. On the other hand in patients with increased levels of plasma creatinine a diminished clearance of N-PAB can be expected. It is also possible that patients without an obvious impairment of liver or renal function may have diminished N-PAB clearance.

This study has been undertaken to investigate the mechanisms of intestinal mucosal transport and metabolism of thymidine analogues and to identify any optimal site(s) of the rat intestine particularly involved in the absorption of thymidine analogues. The intestinal absorption of 3′-azido-3′-deoxythymidine (AZT) was studied at three initial concentrations in four segments of the rat intestine using an in situ recirculating perfusion technique. Disappearance of AZT followed first-order kinetics throughout the gastrointestinal (GI) tract at all tested concentrations. The apparent first-order rate constants were found to be relatively invariant over a broad range of concentrations from 0.01 to 1.0 mM. Corrected for the length of each segment, the apparent permeability (Papp) of AZT was 3.01 ± 0.32 × 10−5 cm/sec (mean ± SE) in the duodenum, 2.06 ± 0.24 × 10−5 cm/sec in the upper jejunum, 0.76 0.13 × 10−5 cm/sec in the combined lower jejunum and ileum, and 0.32 ± 0.10 × 10−5 cm/sec in the colon, which indicated that intrinsic absorptivity was greater in the upper GI tract than in the lower portions possibly due to the differences in surface area for absorption. No AZT metabolite appeared in any part of the GI tract. On the other hand, thymidine and other analogues, i.e., 5-iodo-2′-deoxyuridine and 2′-deoxyuridine, were rapidly metabolized into nucleobase and sugar in the upper GI tract, whereas in the colon no metabolite appeared. A free 3′-OH group appears to be necessary for the metabolism (catabolism) of thymidine analogues in the rat intestine mainly by pyrimidine nu-cleoside phosphorylase. Finally, bile salt-acylcarnitine mixed micelles appeared to be an effective adjuvant in promoting colonic absorptions of AZT and phenol red. The use of mixed micelles increased the apparent permeabilities of AZT in the colon by a factor of 5.4, and for phenol red the permeability increased from a negligible value to 1.76 × 10−5 cm/sec. Since the absorptions of both AZT and phenol red were enhanced by mixed micelles, a paracel-lular transport pathway may be involved.

Summary

After oral and intravenous administration of radiolabelled isobutylnaphthyl acetic acid (INAA) to rats two metabolites were isolated from urine and plasma by HPLC. Field desorption, high resolution electron impact mass spectrometry as well as GC-MS after derivatization were used for structure elucidation and identification of the metabolites. The main biotransformation product in rat urine was found to be 5-(2′-hydroxy-2′-methyl-propyl)-l-naphthyl acetic acid (M1). The main metabolite in plasma was derived and was found to be 5-(2′-carboxypropyl)-l-naphthyl acetic acid (M2).

Summary

Metabolites of tolfenamic acid appearing in human urine have been isolated and their structures determined by C-13 nuclear magnetic resonance and gas chromatography-mass spectrometry. Comparative studies on tolfenamic, mefenamic, and flufenamic acids in conjunction with the metabolites have permitted complete C-13 NMR assignments for this series of compounds. Five metabolites identified included three that were monohydroxylated, one that was both methoxylated and hydroxylated, and another in which the methyl group was oxidized to a carboxyl group. The information presented on the fenamate standards and the metabolites represents an excellent basis for structural elucidation of other fenamates and their metabolites.

Summary

The human metabolism of butobarbitone has been studied in two healthy volunteers at two dose levels, and using sequential dosing. The results are examined graphically, and shown to be consistent with first order processes, provided that due allowance is made for the effects of liver induction. Induction reaches a maximum 3–4 days after a single therapeutic dose; but is still rising after about 10 days, when repetitive doses are given. There is no evidence for saturable kinetics, in the therapeutic dose ranges used in this study.

Summary

Bromocriptine, a D2 receptor agonist, was administered intravenously (1 mg/kg) to anesthetized rats. Microdialysis probes were implanted in the pituitary and the striatum, known sites of D2 agonist action. Bromocriptine and its metabolites were monitored in plasma and tissue dialysates for 4 h. Drug analyses were performed using two different enzyme immunoassays specific for untransformed bromocriptine or a pool of parent drug plus hydroxylated metabolites. The metabolites/parent drug ratio for areas under the curve was 5.5. in plasma and 1 in the pituitary. No metabolites could be detected in the striatum. Bromocriptine penetration was at least 10-fold greater in the pituitary than in the striatum. The kinetics of bromocriptine in the pituitary and striatum did not parallel those in plasma, indicating that the prolonged action of bromocriptine reported by other authors may be due to slow dissociation from receptors.

Summary

The biotransformation of detomidine, a new a2-adrenoceptor agonist, was studied using rat as the model animal. In vivo metabolism of the tritiated drug was compared to in vitro incubations with liver homogenates and intact, isolated hepatocytes. Metabolites were analysed by HPLC with radioactivity detection.

The metabolic patterns in all systems were closely related. HPLC of urine gave twelve radioactive peaks. Tritiated water and unchanged3H-detomidine were minor components. The two major peaks were tentatively identified as hydroxylated detomidine (14%) and its O-glucuronide (43%). Sulphate conjugates were not found.

Isolated hepatocytes converted detomidine to the same two major products; the relative amount of the glucuronide increased with incubation time. In liver post-mitochondrial supernatant, hydroxylation was the dominant reaction, and the hydroxylated product comprised 74% of the total metabolites with non-induced and 50% with phenobarbital-induced liver.

The major biotransformation in rat was thus concluded to be hydroxylation by the liver monooxygenases followed by glucuronic acid conjugation. The maximal rate of oxidation or the enzymatic capacity of a whole liver was estimated to be at least 100 nmol/min allowing for a high hepatic extraction ratio for detomidine. Together with the effective excretion of the glucuronide, this reaction sequence alone could account for the rapid elimination of the drug.